Neofusicoccum cryptoaustrale Pavlic, Maleme, Slippers & M.J. Wingf.

Pereira, Diana S. & Phillips, Alan J. L., 2023, Botryosphaeriaceae on palms-a new species of Neodeightonia, N. chamaeropicola, and new records from diseased foliage of ornamental palms in Portugal, Phytotaxa 627 (1), pp. 1921-1935 : 1921-1935

Neofusicoccum cryptoaustrale Pavlic, Maleme, Slippers & M.J. Wingf. , Persoonia 31: 271 (2013), MycoBank MB512477

( Figure 12 , 13 )

New synonym: Neofusicoccum stellenboschianum Tao Yang & Crous [as “ stellenboschiana ”], Fungal Biology 121: 339 (2017), MycoBank MB840561.

Type: SOUTH AFRICA, Gauteng Province, Pretoria, from branches and leaves of living Eucalyptus trees ( Myrtaceae ), May 2005, H.M. Maleme (holotype PREM 59817, culture ex-type CBS 122813; paratype PREM 59818, culture ex-paratype CMW 20738).

Sexual morph not reported. See Crous et al. (2013) for illustrations and descriptions of asexual morph.

Isolate CDP 1565. Sexual morph: Ascostromata produced on palm leaflets in culture within 2 mo of incubation, pseudothecial, globose to subglobose, with a central ostiole, not papillate, uniloculate, black, solitary or aggregated in small groups, intermingled with conidiomata, immersed in the host with only the ostiolar region emerging, thick-walled, wall comprising several layers of textura angularis, outer region of dark brown or brown cells, inner region of hyaline cells lining the locules. Asci bitunicate, with a well-developed ocular chamber and a prominent axial canal, fissitunicate, clavate, broadly rounded at the apex, stipitate, 8-spored, arising from the base of the ascostromata, formed between pseudoparaphyses, 108–174 × 18–24 μm (including stipe). Pseudoparaphyses cylindrical, smooth-and thin-walled, septate, occasionally slightly constricted at the septum, rarely branched at the base, 2–5 μm wide. Ascospores fusoid to ovoid, ends obtuse to subobtuse, smooth- and thin-walled, hyaline and aseptate, with granular contents, irregularly biseriate in the ascus, (6.95–)15.2–20.07(–22.56) × (3.59–)7.79–10.9 μm, 95 % confidence limits = 16.22–18.46 × 8.12–9.22 μm (mean ± SD = 17.34 ± 4.24 × 8.67 ± 2.08 μm), mean ± SD ascospore length/width ratio = 2.01 ± 0.19 (n = 55). Asexual morph: Conidiomata on palm leaflets in culture pycnidial, morphologically similar to ascostromata, globose to subglobose, non-stromatic, uniloculate, black, solitary, occasionally aggregated in small groups, often intermingled with ascostromata, semi-immersed to superficial or immersed in the host becoming erumpent when mature, densely covered by greyish to olivaceous mycelial hairs. Conidiophores mostly reduced to conidiogenous cells, when present, formed from the cells lining the locule wall, hyaline, smooth- and thin-walled, simple, doliiform to subcylindrical, aseptate. Conidiogenous cells hyaline, smooth- and thin-walled, simple, discrete, integrated, cylindrical to subobpyriform, often tapering towards the apex, straight or curved, aseptate, determinate, holoblastic, often indeterminate, enteroblastic, proliferating at the same level giving rise to periclinal thickenings, or rarely proliferating percurrently to form 1 indistinct annellation, (9.59–)11.77–20.84(–22.32) × 2.2–4.92 μm, 95 % confidence limits = 13.54–15.11 × 3.47–3.75 μm (mean ± SD = 14.32 ± 3.04 × 3.61 ± 0.54 μm, n = 50). Conidia fusiform to oval, often widest in the middle or upper third, base subtruncate to bluntly rounded, apex obtuse to subobtuse, smooth- and thin-walled, hyaline and aseptate, rarely becoming 1-septate before germination, with granular contents and conspicuous guttules, 16.79–25.03 × 4.97–7.47 μm, 95 % confidence limits = 20.19–21.14 × 5.80–6.10 μm (mean ± SD = 20.67 ± 1.81 × 5.95 ± 0.57 μm), mean ± SD conidium length/width ratio = 3.51 ± 0.27 (n = 50).

Culture characteristics: Colonies on 1/2 PDA, reaching 76 mm diam. after 7 d at 20 ℃ in darkness. Surface flat, slightly fluffy, with few aerial mycelia, with filamentous, entire margin, circular shape, whitish to pale, opaque. Reverse pale to yellowish, becoming brownish towards the centre. Turning entirely orangish to olivaceous-grey (surface) and dark brown (reverse) after about 2 w. No diffusible pigment.

Material examined: PORTUGAL, Lisbon, Parque das Nações, on foliar lesions of segments of Chamaerops humilis ( Arecaceae ), 8 May 2021, Diana S. Pereira (specimen HDP 091, new host and geographical record), living culture CDP 1565 (ITS sequence OQ996235, tef1 sequence OR233672, tub2 sequence OR233690); Parque das Nações, on foliar lesions of segments of Chamaerops humilis ( Arecaceae ), 8 May 2021, Diana S. Pereira (specimen HDP 098), living culture CDP 1968 (ITS sequence OQ996242, tef1 sequence OR233676, tub2 sequence OR233691).

Hosts: Arum italicum ( Araceae ), Avicennia marina ( Acanthaceae ), Bruguiera gymnorhiza , Ceriops tagal ( Rhizophoraceae ), Chamaerops humilis ( Arecaceae ), Eucalyptus sp. , E. citriodora ( Myrtaceae ), Lumnitzera racemosa ( Combretaceae ), Malus domestica ( Rosaceae ), Mangifera indica ( Anacardiaceae ), Persea americana ( Lauraceae ), Phoenix canariensis ( Arecaceae ), Podocarpus latifolius ( Podocarpaceae ), Prunus armeniaca ( Rosaceae ), Quercus suber ( Fagaceae ), Rhizophora mucronata ( Rhizophoraceae ), Syzygium cordatum ( Myrtaceae ), Vaccinium corymbosum ( Ericaceae ), Vitis sp. and V. vinifera ( Vitaceae ) ( Farr & Rossman 2023, present study).

Distribution: Algeria, Australia, Portugal, South Africa (including the Eastern Cape, Gauteng, KwaZulu-Natal, Western Cape provinces) and Spain ( Farr & Rossman 2023, present study).

Notes: Neofusicoccum stellenboschianum is reduced to synonymy under N. cryptoaustrale . According to the phylogenetic analyses in this study, isolates of N. cryptoaustrale and N. stellenboschianum clustered together in a monophyletic clade supported with high ML-BS value ( Figure 5 ). The ex-type (CBS 122813) and the ex-paratype cultures (CMW 20738) of N. cryptoaustrale have the following nucleotide similarities with the sequences of the ex-type of N. stellenboschianum (CBS 110864). On ITS: 99.79 % (475/476) and 99.79 % (475/476, including 1 gap), respectively. On tef1: 99.62 % (264/265) and 99.25 % (263/265), respectively. On tub2: 99.74 % (385/386) and 100 % (386/386), respectively. On rpb2: 96.13 % (571/594) and 100 % (594/594), respectively. Based on the phylogenetic analyses of the combined ITS- tef1-tub2 dataset, the strains CDP 1565 and CDP 1968 clustered with the ex-type, ex-paratype and other authentic strains of N. cryptoaustrale with high ML value ( Figure 5 ). Sequence comparisons with the ex-type (CBS 122813) and ex-paratype (CMW 20738) strains of N. cryptoaustrale for ITS, tef1 and tub2 showed 99.37–99.58 %, 98.11–100 % and 99.48-99.74 %, respectively, sequence similarity, and differences were represented by gaps on ITS1, a single base pair change on ITS2 and few base pair changes in both tef1 and tub2 partial sequences. Morphologically, the strains isolated in this study are similar to the holotype of N. cryptoaustrale from branches and leaves of living Eucalyptus trees in South Africa ( Crous et al. 2013) ( Figure 13 ). Considering the strain characterized here (CDP 1565) and the ex-type strain of N. cryptoaustrale (CBS 122813), both produce black, solitary pycnidial conidiomata with fusiform, hyaline and aseptate conidia of relatively similar mean size (20.67 × 5.95 μm and 19 × 5.5 μm, respectively) ( Crous et al. 2013) ( Figure 13 ). Nevertheless, although 1-septate conidia have been rarely observed, CDP 1565 never produced pigmented conidia such as those described for the ex-type strain (CBS 122813) ( Crous et al. 2013) ( Figure 13 ). Thus, based on these morpho-molecular analyses, strains CDP 1565 and CDP 1968 are here reported as representing intraspecific variation of N. cryptoaustrale . Isolate CDP 1565 also produced fertile ascostromata on palm leaflets in culture after 2 months of incubation and thus the sexual morph of N. cryptoaustrale is reported here for the first time. The sexual morph of N. cryptoaustrale is similar to the sexual morph of its sister taxon N. australe ( Phillips et al. 2013) , however the ascospores of N. cryptoaustrale (CDP 1565) are shorter, but wider than those reported for N. australe (17.34 × 8.67 μm versus 21.9 × 7.6 μm, respectively), displaying in consequence a smaller L/W ratio (2.01 versus 2.9) ( Figure 12 ). Neofusicoccum cryptoaustrale have been previously reported on Arecaceae , namely on Phoenix canariensis in Australia ( Cunnington et al. 2007 as Botryosphaeria australis , later ratified by Burgess et al. 2019). However, N. cryptoaustrale has not previously been recorded on Chamaerops humilis and is reported for the first time from Portugal, representing a new host and geographical record ( Table 5). The isolates of N. cryptoaustrale studied here were recorded from foliar lesions of C. humilis , but pathogenicity has not been tested.

Figures

FIGURE 5. Phylogenetic tree of Neofusicoccum generated from BA inference based on combined ITS, tef1 and tub2 sequences. ML bootstrap support values (ML-BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes. Strains with type status are indicated in bold font, the isolates from this study are presented in brown typeface and species are delimited with coloured blocks. Species names in N. parvum species complex are noted before the strain number. The scale bar represents the expected number of nucleotide changes per site. The tree was rooted to Botryosphaeria corticis (CBS 119047) and B. dothidea (CBS 115476).

FIGURE 12. Sexual morph of Neofusicoccum cryptoaustrale (CDP 1565). A, B. Asci intermingled with pseudoparaphyses. C. Pseudoparaphyses. D–F. Immature (D, E) and mature (F) asci. G, H. Details of ascus apex. I–N. Ascospores. Scale bars: A, B, D–F = 10 μm, C, G–N = 5 μm.

FIGURE 13. Asexual morph of Neofusicoccum cryptoaustrale (CDP 1565). A–C. Conidiogenous layer. D–I. Conidiogenous cells. J, K. Conidia. Scale bars:A–C = 5 μm, D–I = 2.5 μm, J, K = 10 μm.